我们感谢加斯帕Kronhamn和丹Hultmark，迈克尔Galko酒店和布卢明顿股票中心飞股票。特别感谢康特尼 - 小野寺咨询与统计分析。我们感谢卡特里娜黄金手稿的批判性阅读和卡尔帕纳·麦克贾尼，卡特里娜黄金的Derynck实验室的成员，以及Nystul实验室进行讨论和评论的手稿的成员。这项工作是由加州大学旧金山分校计划赠款突破生物医学研究（PBBR），远大中心，赫尔曼基金会，美国癌症协会RSG DDC-122595，美国心脏协会13BGIA13730001，美国国家科学基金会1326268，卫生1R01GM112083-01全国学院和支持1R56HL118726-01A1（以KB）。

1，血细胞出血/刮分析<ol><li>准备幻灯片： <ol><li>选项​​1的显微镜没有瓦片扫描功能：对于每一个幼虫要分析，制备一种玻璃载玻片用各2毫米的正方形约5巴氏笔井，对应于显微镜的领域观察区域;添加约5 - 10微升S2媒体的每个（图1A）。保持滑动保湿室，以防止井干燥。 </li><li>选项​​2为显微镜用瓷砖扫描功能：对于每一个幼虫待分析，准备为〜3 3至4巴氏笔井1载玻片 - 每4平方毫米;加约15 - 20微升S2介质到每个孔中（图1B）。保持滑动保湿室，以防止井干燥。 注：孔的上述建议的数量是足够的每幼虫总数高达3000血细胞（后第二龄幼虫，〜2.5 - 3毫米的长度，转基因标签的大部分larv的人血细胞）。当评估较大血细胞数目，更多的井可能需要避免过度拥挤。 </li></ol></li><li>收集幼虫： <ol><li>喷射水成含有幼虫和冲洗幼虫到培养皿蝇小瓶，或舀一些食品包含幼虫到培养皿，并用喷瓶，用水稀释。 </li><li>轻轻一挑出来的幼虫用画笔培养皿中，并放置在水中一腔的菜，或在冷块上的幻灯片。 注：幼虫可以保持在水或冷块上的有限的时间;使用在45分以下的标本，以免幼虫死亡或血细胞有害的影响。 </li></ol></li><li>解剖： <ol><li>冷金属块在荧光显微镜下选择幼虫。如果需要测量尺寸和图像幼虫。 </li><li>隔离循环血细胞（“渗透”）的： <ol><li>一旦幼虫被选择时，将一只幼虫在第一巴氏笔井（图1C，2A）。 </li><li>使用2清洁针具或解剖剪刀和镊子，使切口同时在后部和前幼虫的末端。为了避免干扰居民的血细胞，最好是在幼虫腹侧使这些切口。对于一致的结果，使每一个幼虫相同位置的切口。为1日龄幼虫，1切口（在腹侧前）就足够了。 </li><li>允许幼虫流血了几秒钟，没有任何压力或身体搅拌（ 图2A）。 注：如果处理多个幼虫，最好在进行下一步骤，以避免保持幼虫在冰上太长可能影响样品“完整性之前，使这些切口为每一个。 </li><li>轻轻抬起用针或镊子幼虫蘸到第二口井，以冲洗任何剩余的循环血细胞。在此之后，按照与居民血细胞释放。 </li></ol></li><l我&gt;居民血细胞分离（“刮”）： <ol><li>幼虫轻轻转移到下一个孔（图2C）。 </li><li>识别幼虫，其典型地位于约1/3从幼虫的前端的淋巴腺，并且其可以通过幼虫体壁背侧荧光。避免淋巴腺，同时通过牵制的幼虫用针尽可能接近到淋巴腺，以避免刺穿（ 图2C），释放居民的血细胞。 注：在正常发展的淋巴腺血细胞的成熟相比，幼虫的血细胞被延迟，并有区别的血细胞的荧光记者可能不会显示在低龄幼虫的淋巴腺的信号。在这些情况下，关注较少需要由淋巴腺血细胞支付给淋巴腺因为没有污染分化荧光标记的幼虫血细胞的预期。 </li><li>在dissecti释放居民的血细胞拼抢和/或猛刺的过程。使用一个针尖根据需要有效地牵制附近的淋巴腺幼虫（见上文）或其他身体部位。使用其他针戳在血细胞可见的通过幼虫体壁（ 图2C，E）的集群，目标是分开的血细胞。血细胞也可以被释放在一个刮运动。然而，早期的撕裂表皮可能释放出的血细胞，这可能使自动计数更具挑战性的大型集群。 注意：根据幼虫的年龄和基因型，总血细胞的数量将有所不同。分发上述过几口井，以避免一些井血细胞，这可能使单细胞图像分析的难度拥挤的释放过程。 </li><li>如果几个血细胞留在最后胴体，算上这些血细胞通过观察通过手动式计数器（ 图2E）的显微镜和使用。为了方便计算，P花边胎体上相同幻灯片的洁净区和传播它尽可能地薄，以减少光学面的数目。 </li><li>一旦解剖完成后，等待之间5 - 10分钟使细胞成像孔之前沉淀（但不一定附着）。孵育幻灯片的保湿室，以避免干燥，避免野蛮装卸的幻灯片，这可能会扰乱安定血细胞。 注：当确定血细胞数量，释放的细胞是不固定的，细胞必须被成像解剖后不久，优选在从幼虫释放后30分钟。 10分钟，这应该通过聚焦通过在阱的介质的光学平面的确认 - 视的介质和电池特性在卷上，将细胞中的绝大多数将已在5结算。但是，血细胞只有一小部分将已经粘附至所述滑动表面通过这个时候，也需要考虑的事实，如果修改这一协议，进行细胞固定基approache秒。 </li></ol></li></ol></li><li>定量： <ol><li>就拿落户血细胞图像的荧光显微镜（ 图2B，D，F）下。按照与使用ImageJ软件血细胞的定量。 </li><li>准备图像ImageJ的细胞计数算法： <ol><li>采用国内的ImageJ打开图像：文件→打开→（找到文件，并选择）。 </li><li>确保图像（S）是8位或16位。调整阈值的图像通过选择图片 ，然后单击调整 ，然后选择阈值 。遵守“ 阈值窗口 ”（ 图3A）。 </li><li>检查“黑暗背景”选项。选择“红色”，并增加了低阀值 （见黑色箭头），直到图像中的每个细胞都标有一个红点（即不被覆盖将灰阶可以看到细胞; 图3B）。作为下限阈值增加某些细胞会变得无标记。这对于多远设置下限阈值指示器。 注：有时细胞簇不能得到解决，将被计为一个由粒子计数器。在这种情况下，细胞在一个簇的数目可以通过检查图像（如果需要放大），并使用式计数器人工计数来估计。或者，所述下限阈值可以提高到解决细胞簇;从该操作产生的任何未标记的细胞然后可以使用一式计数器计数。 </li></ol></li><li>使用ImageJ分析细胞数： <ol><li>发射粒子分析仪计数细胞 （图3C）。选择分析 ，然后单击分析粒子。（可选）选择“叠加纲要 ”中看到的粒子计数算法（图3D）。可替代地，设置限制向一个单元的大小或像素的区域（ 例如。，细胞， 细胞等）的算法的丛计数。 </li><li>单击确定。观察与计数（ 图3E）的摘要窗口。 </li></ol></li></ol></li></ol> 2.血细胞干扰试验<ol><li>打扰血细胞，选择幼虫，并将其放置在一个2 ml离心管约为0.5克玻璃珠（212 - 600微米），并加入0.5毫升的水。 </li><li>涡管，通过手，以速度10℃1分钟。 </li><li>由溢出的微量离心管中的内容到培养皿并挑选出幼虫用画笔检索来自玻璃珠的幼虫。 </li><li>为恢复阶段，放置在幼虫预先制备培养皿与少量飞食品。允许幼虫以重新建立它们的血球图案的一段45分钟或根据需要。 注：放弃已经停止运动，因为他们在这个过程中已经死亡的任何幼虫。然而，我们通常见升后ittle损害涡旋1分钟（见下文和补充图1）。 </li><li>恢复期后，继续出血/刮含量，如第1如上所述。 </li></ol>

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The authors have nothing to disclose.

这里，我们描述定量从单个果蝇幼虫回收居民和循环血细胞，并量化这两个血细胞种群的第一个方法。该协议包括下列顺序释放循环和驻地的血细胞中，接着成像和自动细胞计数。幼虫居民血细胞可瞬时动员到循环由机械干扰，已知能够在很大程度上30内反转的处理- 60分钟恢复期6。因此，该协议以两种方式进行测试，（1）通过评估每幼虫和循环血细胞过度幼虫发育的过程中的分数的总血细胞数，和（2）通过使用自动化方法实验撞出驻地血细胞，这证实了血细胞定位和驻地血细胞数量和循环群体紧密的联系。另外，该方法的再现性所表明的共mparing生物学重复的两个数据集。 在过去，实验室已使用一系列的技术来量化幼虫血细胞6,13,21。该协议确立了一个共同的标准来检索和果蝇幼虫量化居民和循环血细胞群，提供了一个容易适应的平台。所描述的方法是用于研究，着眼于居民血细胞和其微环境的作用至关重要，造血口袋4-6，并适合于研究荧光蛋白基因携带果蝇品系在野生型和转基因的背景。该协议也是相关的研究，着眼于血细胞动员免疫的挑战和伤害，以及遗传或环境引起的信号触发动员居民血细胞或改变总血细胞数（在4审查）之后。应当指出的是，在二过早的情况下fferentiation和从淋巴腺血细胞的释放，区分胚胎/幼虫与淋巴腺谱系可通过使用的荧光报道血细胞的表达模式来限定。 这里介绍的协议依赖于成像活，荧光标记的血细胞。在将来，它可被修改，以允许释放的细胞的检测固定后， 例如，使用免疫细胞化学。在这种情况下，协议可能需要被调整，以确保血液细胞完全粘附，例如通过增加粘附孵育时间，和加入粘合剂滑动涂层，例如伴刀豆球蛋白A.由于该方法允许血细胞的检索和其操作前体内 ，这将有利于广泛发育，细胞生物学和生物化学的研究。在果蝇和所有胚后发育阶段其他无脊椎动物22，suggesti发现居民和循环血细胞NG这种方法，适应将有利于广泛的超越了果蝇幼虫造血系统的研究。

研究中的无脊椎动物模型果蝇驱使先天免疫1的发现，并且已经促进了血液细胞发展的各个方面的理解2-4。 果蝇造血可分为胚胎/幼虫的血细胞谱系，其起源于胚胎和扩大的幼虫，以及淋巴结的血细胞血统4,5。在这里，我们提出了一个协议，侧重于胚胎/幼虫血细胞谱系，这在果蝇幼虫主要由浆细胞（巨噬细胞）和少量晶体单元4。位于表皮和幼虫体壁5,6的肌肉层之间的幼虫，胚胎血细胞的坚持和殖民节段性重复和终端造血口袋（HPS）。根据其性质，自我更新的巨噬细胞6，局部组织微环境6的主要居住地，从最早的血细胞发育6,8-期间新兴7，和它们的谱系，这血细胞种群被认为类似于脊椎动物自我更新的组织巨噬细胞，一个独立的骨髓系最近在各种物种4,9,10的鉴定。然而，在果蝇中，一些或所有这些常驻细胞也显示可塑性以产生以下的其他血细胞类型如晶单元11,12。 幼虫血细胞主要是驻留（无梗），但在不同的惠普之间的动态稳定状态。它们被逐步释放进入循环，特别是作为第 3龄幼虫接近pupariation 5-7。免疫挑战，受伤或机械干扰导致过早，在后者的情况下是可逆的，动员居民血细胞的成血淋巴4,6,13。 以往的研究表明，居民和循环幼虫血细胞是一脉相承的，但会有不同的粘合剂或归巢性-6,7,13,14。循环与居民血细胞的选择性分离透露增殖水平升高在驻地血细胞的人口，这表明它们暴露于高压钠灯6。 果蝇幼虫惠普感性线索由表皮和肌肉层排列 ​​，并进一步海港周围神经系统的感觉神经元集群（PNS）和肝功能类似oenocytes 6。在功能上，突变和遗传细胞消融实验表明，目前在惠普感觉神经元支持营养存活幼虫血细胞6和定位。 这里，我们描述了具体分离和居民的定量和从单一果蝇幼虫循环血细胞，以及协议为机械血球动员的方法。该方法可用于体外血细胞的研究，并且可以进一步适于诸如蛹和成虫，以及其他无脊椎动物系统其他果蝇发育阶段。由于先前的研究没有居民，循环血细胞区分，该协议规定了居民的血细胞的研究中一个共同的标准，将有助于提高无脊椎动物的血细胞研究的一致性。 首先， 血细胞出血/刮测定描述差分隔离和荧光蛋白标记的居民，循环从单一的果蝇幼虫血细胞种群的自动定量;该协议提供了两种经常和瓷砖扫描配备显微镜（ 图1）。其结果，得到的循环血细胞的百分比和每幼虫血细胞的总数。该方法依赖于表达他们当中的血细胞荧光蛋白转基因果蝇幼虫人口。血球司机或报道的选择将决定结果， 即，该血液细胞群被可视化和量化。主要标记的巨噬细胞（浆细胞），其中包括居民和果蝇幼虫6的循环血细胞人口的绝大多数，合适的转基因包括HMLΔ-红色荧光蛋白6，HmlΔ-GAL4 15，PXN -GAL4 16，CRQ-GAL4（由H. Agaisse 16），或吃-GAL4 17;用于标记的人口相对较少的晶体电池，适合线BcF6-CFP和-GFP 18，或LZ-GAL4（由J.波洛克19）;用于标记lamellocytes，一个专门的细胞类型主要是诱导免疫的挑战和伤害13， 例如，MSNF9mo-mCherry可以使用17。一些转基因的驱动中表达的范围的分化血细胞和祖细胞，如他 - GAL4 20，其中标签约80％的幼虫血细胞20。请注意，在所有的情况下，其中的GAL4的驱动时，与UAS-GFP或另一荧光蛋白UAS -转基因的组合是必需的。在结果部分 ，这种方法用于监测血细胞数量和流通行为在幼虫发育的过程。 第二， 血细胞干扰测定描述旨在通过外部操纵，其随后允许血细胞重新坚持和家庭惠普在有限的时间范围内的能力的评估，以分离驻地血细胞的在先步骤（30 - 60分钟）6。典型地该测定后跟出血/刮除试验，以确定每幼虫循环血细胞的百分比。我们提出了一个简化的协议，用于该测定法（图1D），其使用扰动通过涡旋机智^ h玻璃珠，而不是操纵与油漆刷单幼虫如前面6所述。 在结果部分，该测定是用来证明瞬时分离的血细胞浮在血淋巴，并且可以在循环血细胞的级分中回收。该测定法也是有用的量化血细胞在其归巢/粘附到驻地站 ​​点的差异，比较例如，各种遗传背景或刺激条件。请注意，该机械操作反映了一个可逆的过程，是从infection-或损伤诱发驻地血球动员，这通常是不可逆的，在短时间内帧4,13不同。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>6cm/9cm Petri dishes</td>
			<td></td>
			<td></td>
			<td>One for each genotype to be evaluated</td>
		</tr>
		<tr>
			<td>Water squirt bottle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal spoon/spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin paintbrush</td>
			<td></td>
			<td></td>
			<td>e.g. a &#34;liner&#34;</td>
		</tr>
		<tr>
			<td>Glass cavity dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen: Super PAP PEN IM3580</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td></td>
			<td></td>
			<td>Each slide will have 5 or more PAP PEN squares drawn on them. Size of squares depends on the imaging objective and magnification of the microscope camera; e.g. 2mm squares.</td>
		</tr>
		<tr>
			<td>Moist chamber</td>
			<td></td>
			<td></td>
			<td>This will be used to prevent slides and wells from drying out: sealed container with wet paper towels lining the sides/bottom</td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila cell culture media</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold block</td>
			<td></td>
			<td></td>
			<td>This is a metal block (a.k.a. heating block) chilled in bucket containing ice; preferably black-colored or other dark, non-reflective color</td>
		</tr>
		<tr>
			<td>Two 1ml syringes with needles (27G &#189;)</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>For dissections.</td>
		</tr>
		<tr>
			<td>Optional: Surgical spring scissors (cutting edge 2mm)</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, 212-600 micron</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>One per genotype evaluating</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transgenic Drosophila larvae with fluorescently marked hemocytes. Suitable transgenes include: Hml&#916;-DsRed (Makhijani et al., 2011), MSNF9mo-mCherry (Tokusumi et al., 2009), BcF6-CFP and -GFP (Gajewski et al., 2007), or Hml&#916;-GAL4 (Sinenko and Mathey-Prevot, 2004), Pxn-GAL4 (Stramer et al., 2005), He-GAL4 (Zettervall et al., 2004), Crq-GAL4 (by H. Agaisse (Stramer et al., 2005)), or eater-GAL4 (Tokusumi et al., 2009) combined with UAS-GFP or other fluorescent protein transgenes.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence dissecting microscope</td>
			<td>Leica</td>
			<td></td>
			<td>Here: Leica M205, optional with camera, imaging software and measuring module</td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope with camera attachment</td>
			<td>Leica or Keyence</td>
			<td></td>
			<td>With or without tile scanning function (eg. Leica DMI series, Keyence BIOREVO BZ-9000 series)</td>
		</tr>
	</tbody>
</table>

assaying blood cell populations of the drosophila melanogaster larva

在脊椎动物中，造血由感应微环境（利基市场）监管。同样地，在无脊椎动物模型生物体果蝇 ，称为幼虫造血口袋（HPS）感应的微环境已被确定为解剖学部位的发展和调节的血细胞（血细胞），特别是自我更新的巨噬细胞系的。惠普是分段地重复幼虫的表皮和肌肉层，其还包含外周神经系统的感觉神经元之间的口袋。在幼虫，驻留（无梗）血细胞暴露于抗细胞凋亡，粘合剂和由这些感觉​​神经元增殖性线索和高压钠灯的潜在的其他部件，如内衬肌肉和上皮层。在正常的发展，居民的血细胞逐渐释放从惠普燃料循环血细胞的人口，达到高潮在莫释放ST驻地血细胞在变态的开始。免疫攻击，人身伤害或机械扰动触发过早释放居民的血细胞进入血液循环。居住场所和循环之间幼虫血细胞的开关提出了需要一种通用的标准/过程以选择性地分离和从单一果蝇幼虫量化血细胞的两个群体。因此，本协议描述了一个自动化的方法来释放和量化，从单一的幼虫驻地和循环血细胞。该方法有利于体外方法，以及可适于提供各种果蝇和发育阶段其它无脊椎生物。

为了说明所描述的方法的典型的结果，我们首先使用了血细胞渗/刮测定勾勒幼虫血细胞数量的进展，他们的住所以上幼虫发育的过程中（图4）。居民和循环幼虫血细胞群体是从单一的幼虫中分离（HMLΔ-GAL4，UAS-GFP;他-GAL4标签 使用ImageJ绝大多数幼虫血细胞）和量化。幼虫队列尺寸1.2毫米（〜48小时AEL或1日龄），2.5毫米（〜80小时AEL或2晚第二龄），和3.5毫米（〜96小时AEL或第三龄）进行了检查（图4） 。血细胞数目扩大了幼虫发育的过程中，与相 ​​关并超过以前的估计基于染料染色幼虫7和荧光蛋白标记的血细胞通过幼虫角质层6个现场计数的光学显微镜。在1日插件焦油幼虫几乎所有的血细胞均居于，而循环血细胞的比例逐渐增加了幼虫发育的过程中（ 图4B，C），与以前的出版物6,7一致。 接下来我们检查方法是否忠实地监视居住和流动人口之间血细胞的过渡。以全球化的现象是居民血细胞可以瞬时通过机械扰动脱落，他们重新坚持自己的居住场所自发6，我们通过与作为血细胞干扰测定法中玻璃珠涡旋分散居住血细胞。的确，幼虫机械干扰导致在牺牲居民血细胞（图5）循环血细胞的人口的急剧增加。经过45分钟的恢复期，血细胞已基本回到了自己的附着状态，无论是通过视觉观察，并由作为循环细胞sessed百分比（图5D 中的E）。正如预期的那样，总血细胞数量保持稳定随着时间的推移，尽管血细胞的循环和居民之间的转变。 另外还有几个因素考虑在内。为了证实涡旋没有引起大的组织损伤，与玻璃珠是在台盼蓝（Sigma）的的不同时间（1,5，20分钟）的存在下进行涡旋。既1和5分钟涡旋没有引起任何明显的组织破坏，而20分钟涡旋导致损害的小区域，类似引起的用作阳性对照（ 补充图1）针针损坏。而表皮或不角质层损伤其它组织的内部破坏，不能排除，这种情况下，似乎相当不可能如1分钟，5分钟处理过的幼虫重新附着在预期图案与时间帧血细胞，提示幼虫完整性不妥协D（ 参考图1）。与此相反，幼虫涡旋20分钟从缺乏再附着遭遇，并没有显示出甚至附着循环血细胞表皮伤口位点，如先前已描述的14。 最后，以证明该方法的再现性，我们比较了2.5毫米幼虫从以上两个实验，这是由不同的实验者进行生物学重复。所示补充图2中，两个队列表明可比总数每幼虫血细胞，和循环血细胞的百分比。学生t检验显示，无统计学差异显著，这表明该方法是可重复的和广泛适用的。 <img alt="图1" src="/files/ftp_upload/52733/52733fig1highres.jpg" width="500" /> 图1.血细胞出血/擦伤和干扰分析小号<html> etup和原理图。 （一）单图片幻灯片设置：五2毫米正方形成像与5倍的目标（B）磁砖扫描幻灯片设置：四3mm见方的成像出血/中≤2.5毫米幼虫瓷砖扫描显微镜擦伤。用于成像的推荐目标是5X或10X。（C）的渗出/使用ImageJ刮测定示意和所得量词。（D）在干扰测定，所述血球图案被机械地通过涡旋幼虫用玻璃珠破碎。幼虫被允许恢复历时45分钟在此期间，血细胞重新粘附到造血口袋。血细胞的粘附特性可以通过这种方法进行评估，量化血细胞中循环障碍后的百分比。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 方法“&gt; <img alt="图2" src="/files/ftp_upload/52733/52733fig2highres.jpg" width="700" /> 图2.出血/刮测定释放循环和居民血细胞。 （A）流血幼虫，腹部切口在幼虫的后部和前部末端均采用（剪刀符号）。（二）血细胞在流通将流出切口，解决了滑动表面上。（C）淋巴腺（LG）位于和牵制，没有刺破它。居民血细胞释放被戳和/或刮用针幼虫。幻灯片（四）居民血细胞（E，F）反复刮过程，直到所有常住血细胞释放。包含完整的淋巴腺的幼虫尸体被抛在后面。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.自动使用ImageJ血细胞的定量。 （A，B）中的ImageJ打开血球图像文件后， 下限阈值电平被调整，以考虑所有的细胞的图像中。（C，D）分析 颗粒需要设定该单元像素尺寸，圆度，且结果读出格式 （例如，覆盖纲要 ）。（E）摘要窗口显示血细胞的数量。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52733/52733fig4highres.jpg" width="700" /> 图4。代表结果（1）。血细胞数量和居民状态经过了<html>当然幼虫发育。 （A）中的幼虫阶段的概述; 1日龄（48小时AEL;约1.2毫米的长度）; 第二届龄（80小时AEL 2.5毫米的长度）; 3 路龄 （96小时AEL;〜3.5级毫米的长度）。基因型是HMLΔ-GAL4，UAS-GFP;他-GAL4。阶段被评估幼虫mouthhooks证实，在各自的幼虫阶段循环和居民血细胞数（B）条形图。循环血细胞（C）的百分比。注意，循环血细胞的分数超过幼虫发育的过程中增加不相称。（D）的总血细胞，从每幼虫循环和驻地血细胞的总和所得。使用血细胞渗出/刮法进行量化; ñ≥6幼虫/条件，误差线显示标准差，结果在3个独立的重复实验证实。JPG“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/52733/52733fig5highres.jpg" width="700" /> 图5.代表性的成果（2）。对影响血细胞居住的机械干扰。 （AC）之前和用玻璃珠，随后45分钟恢复涡旋后幼虫的实施例（A）的无干扰控制。血细胞在玻璃珠和水中的悬浮液涡旋幼虫后定位于造血口袋（B）的血细胞打乱模式，在0 分钟（℃）在恢复后的干扰45分钟血球图案。;许多血细胞已经搬迁到造血的口袋里。注意放大背血管相关的集群和腕背横纹这是早期干扰后的积累（箭头）为主的网站。基因型是HMLΔ-GAL4，UAS-GFP;他-GAL4 x YW。循环血细胞的出血/刮法进行定量的（D）的百分比。（E）的总血细胞，每幼虫流通和居民血细胞的总和产生的。 ñ≥4幼虫/条件，误差线显示标准差，结果在3个独立的重复实验证实。学生的t检验，以确认意义，NS（不显著），**（P≤0.05），**（P≤0.01）。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Assaying Blood Cell Populations of the Drosophila melanogaster Larva at JoVE.com

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

assaying-blood-cell-populations-of-the-drosophila-melanogaster-larva

Research

JoVE Journal

Developmental Biology

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

12.2K 次观看.  University of California San Francisco. Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

视频: Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

科研

发育生物学

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...